1. Field of the Invention
This invention relates to a method and apparatus for the measurement of antigens and antibodies. More particularly, this invention relates to a method of the quantitative measurement of antigens and antibodies by supporting an antibody or an antigen on insoluble carrier particles having minute particle diameters to sensitize the insoluble carrier particles, then reacting the sensitized carrier with a corresponding antigen, antibody or mixture thereof and irradiating the reaction mixture with light of a specific wavelength to measure the transmitted light at two or more points of time, and then evaluating an increase in absorbance or percent absorption of the reaction mixture for a given period of time, and an apparatus for use therein.
2. Description of the Prior Art
There is a continuing need for rapid, accurate, qualitative and quantitative determinations of biologically active substances, e.g., antigens, antibodies, at extremely low concentrations. Today, there is a wide need for determining the presence of drugs in body fluids. In addition, in medical diagnosis, it is frequently important to know the presence of various substances which are synthesized naturally by the body or ingested.
Heretofore it has been known to detect antibodies or antigens semiquantitatively by reacting latex particles on which an antibody or an antigen is supported with a corresponding antigen or antibody on a glass plate and observing visually the agglutination state.
In recent years, it was proposed in the following articles to quantitatively determine antigens and antibodies using the above-mentioned latex particles by supporting the corresponding antibody or antigen on the latex particles to sensitize the latex, reacting the supported antibody or antigen with the antigen or antibody to be determined to agglutinate the latex particles, and measuring the rate of decrease in turbidity of the supernatant of the latex by means of visible lights for the determination of the antigen or antibody utilizing the agglutination phenomena of the latex reagent:
(A) CROATICA CHEMICA ACTA, 42, (1970), p.p. 457-466; and PA0 (B) European Journal of Biochemistry, Vol. 20, No. 4, (1971), p.p. 558-560. PA0 (C) Immunochemistry, Vol. 12, p.p. 349-351 (1975)
Since the method of the above proposal utilizes the measurement of rate of decrease in turbidity to determine the antigen or antibody, it is necessary to use an antibody- or antigen-sensitized latex of an extremely low concentration, for example, in the range of 0.007 to 0.028%, to carry out the reaction of the latex and the antigen or antibody in a stationary state, to remove any impurity capable of affecting the turbidity from the sample to be tested, and the like. As a result, the above-mentioned method is disadvantageous in that the rate of the antigen-antibody reaction is inevitably decreased, both the precision and the reproducibility are insufficient for the determination technique of antigens or antibodies, and that the removal of impurities sometimes requires extremely complicated operations. Accordingly it is difficult to apply the above method to the determination of such antigen as fibrinogen (Fg), human chorionic gonadotropin (hCG) or the like, since it requires complicated procedures for the preparation of its reagent and it is difficult to cause reproducible agglutination reaction if it is present in blood or urine which also contains various other substances capable of adversely affecting the reaction.
Also in the article,
it was proposed to determine quantitatively antibodies and antigens by irradiating the above-mentioned agglutinated latex particles with a laser beam and measuring the change in width of spectral lines of the scattered light of the laser beam in order to determine the mean diffusion constant (D) which gives an indication of the Brownian motion of the agglutinated particles which in turns is inversely proportional to the size of the agglutinated particles. Also in this method, since the antibody- or antigen-sensitized latex is used in an extremely low concentration, for example, as low as 0.001%, the rate of the antigen-antibody reaction is so decreased that both the precision and the reproducibility become poor. In addition, this method is also disadvantageous in that it requires complicated calculation using the technique of spectrum analysis which in turn requires complicated operations, and that any impurity in the sample must be removed prior to the measurement. Accordingly, this method has not been put into practice as well.
The above paper C also describes that determination by the turbidity method as reported in the foregoing paper A gives extremely imprecise results (FIG. 2 on page 350 of the same).
As a result of our investigation with respect to methods and apparatus for the rapid determination of an antigen and/or antibody in a sample to be tested with a high precision and a good reproducibility, we formerly accomplished an invention which is the subject matter of Japanese Patent Application No. 97158/76 (hereinafter referred to as "our prior filed application").
The invention of our prior filed application above resides in a method for measuring antigens and antibodies comprising supporting an antibody or antigen corresponding to the antigen or antibody to be determined on insoluble carrier particles having an average diameter of not greater than 1.6 microns, reacting the supported antibody and/or antigen with the antigen or antibody or a mixture thereof to be determined and irradiating the reaction mixture with light having a wavelength in the range of 0.6 to 2.4 microns and longer than the average diameter of said carrier particles by a factor of at least 1.5 to determine the absorbance of the reaction mixture.